McLean S, Rothman R B, Chuang D M, Rice K C, Spain J W, Coscia C J, Roth B L
Laboratory of Neurophysiology, NIMH, Bethesda, MD 20892.
Brain Res Dev Brain Res. 1989 Feb 1;45(2):283-9. doi: 10.1016/0165-3806(89)90046-1.
Radioiodinated human beta-endorphin was cross-linked to opioid receptors from rat brain membranes using the bifunctional reagents bis-[2-(succinimidooxycarbonyloxy)ethyl] sulfone (BSCOES) and disuccinimidyl suberate (DSS). Major radiolabeled bands migrated with Mr values of 65,000, 55,000 and 33,000, however the presence of the 55 kDa band was variable. The 65 kDa band was characterized as the mu-receptor: the binding of [125I]beta-endorphin to this band was displaced by mu-selective ligands and blocked by alkylation of the receptor by mu-specific, but not delta-specific alkylating agents. The cross-linked receptor underwent alterations in mol. wt. during development. Early in development, embryonic day 18 and postnatal day 1, the [125I]beta-endorphin-labeled material migrated as a single band of mol. wt. 55 kDa. By day 21 postnatally the higher mol. wt. band of 65 kDa was present, as was material of 53, 47 and 43 kDa. Although the protein labeled early in development migrated with a mol. wt. of 55 kDa similar to the delta-receptor isolated from NG108-15 neuroblastoma-glioma cells, competition studies suggest this protein is not the delta-receptor. The 65 kDa band, tentatively identified as the mu-receptor, was present in adults but not detected in neonates, despite competition binding data indicating the presence of mu-sites. The results suggest that the 55 kDa band found in the 1-day-old neonate may be an immature form of the mu-opioid receptor that undergoes posttranslational modification, perhaps glycosylation, during development.
利用双功能试剂双-[2-(琥珀酰亚胺氧基羰基氧基)乙基]砜(BSCOES)和辛二酸二琥珀酰亚胺酯(DSS)，将放射性碘化人β-内啡肽与大鼠脑膜中的阿片受体交联。主要的放射性标记条带迁移时的分子量分别为65,000、55,000和33,000，然而55 kDa条带的存在情况并不稳定。65 kDa条带被鉴定为μ受体：[125I]β-内啡肽与该条带的结合可被μ选择性配体取代，并被μ特异性而非δ特异性烷基化剂对受体的烷基化所阻断。交联后的受体在发育过程中分子量发生了变化。在发育早期，胚胎第18天和出生后第1天，[125I]β-内啡肽标记的物质迁移为一条分子量为55 kDa的单一谱带。出生后第21天，出现了分子量更高的65 kDa条带，以及53、47和43 kDa的物质。尽管在发育早期标记的蛋白质迁移时分子量为55 kDa，与从NG108-15神经母细胞瘤-胶质瘤细胞中分离出的δ受体相似，但竞争研究表明该蛋白质不是δ受体。初步鉴定为μ受体的65 kDa条带在成年动物中存在，但在新生儿中未检测到，尽管竞争结合数据表明存在μ位点。结果表明，在1日龄新生儿中发现的55 kDa条带可能是μ阿片受体的未成熟形式，在发育过程中经历了翻译后修饰，可能是糖基化。
